Natural Products as Pharmacological Tools to probe Mitochondrial and Secretory Function in Cancer Cells

Cancer is the second leading cause of death in the United States. To date, over 60% of current anticancer drugs were inspired by the discovery of chemical structure from nature, commonly known as a natural product. This work represents the biological characterization of several marine natural product compounds and their use as pharmacological tools to study mitochondrial and secretory functions in human cancer cells. The natural product mandelalide A was recently found to be a direct inhibitor of ATP synthase. We revealed that mandelalide A induces transient activation of the AMP-activated protein kinase pathway in an LKB1-dependent manner in several human cancer cells. Mandelalide A induces a synergistic affect in combination with first-line antitumor agents erlotinib and paclitaxel on EGFR mutant non-small cell lung cancer cells. Another new class of cytotoxic marine natural products, coibamide A and apratoxin A, were found to use autophagy-related protein 5 (ATG5) to promote cell death signaling. Mouse embryonic fibroblasts (MEFs) with a functional autophagy pathway were more sensitive to these rare natural product compounds which are inhibitors of the secretory pathway. The secretory pathway is a potentially novel, and future target for cancer therapy. We developed a workflow for discovery of new secretion inhibitors by combining a functional Gluc secretion assay with recognized cell viability assays. We used this workflow to identify 6 small molecules out of 3906 screened, and characterized the inhibitory activity of coibamide A in this screen. Using a cellular thermal shift assay, we identified the translocon protein Sec61 alpha as a direct binding target of coibamide A. We discovered that the resident endoplasmic reticulum (ER) chaperone protein glucose regulated protein 78 (GRP78) is a critical, indirect target of coibamide A. High basal expression of GRP78 and distribution to cellular compartment outside the ER has been increasingly associated with human cancers. We identified upregulation of GRP78 expression in canine osteosarcoma cells and investigated the use of an anti-cancer drug OSU-03012 to target GPR78 overexpression in these cells. In summary, we concluded that activation of AMPK pathway by mandelalide A-like compounds, and inhibition of secretory function or secretory client proteins by coibamide A-like compounds could be beneficial as therapeutical strategies in cancer treatment

Adiponectin exhibits proliferative and anti-apoptotic effects on ovarian cancer cells via PI3K/Akt and Raf/MEK/ERK pathways

Purpose: To elucidate the effects and the underlying mechanism of adiponectin on human ovarian cancer cells.Methods: The level of adiponectin, adiponectin receptor-1, caspase-3 and bcl-2 in the serum and ascites of the patients were measured with enzyme-linked immunosorbent assay (ELISA), qPCR and Western blotting. The human ovarian cancer cell lines (Caov3 and SKOV3) were enumerated using 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium bromide (MTT) assay. Western blotting was also used to determine the levels of p-Akt, p-ERK and cyclin B.Results: Serum and ascite levels of adiponectin were significantly higher in ovarian cancer patients than in healthy patients (p < 0.05). Expression of adiponectin in the serum and ascites of patients in FIGO stage IV was remarkably higher than in earlier stages (p < 0.05). The proliferative effect of adiponectin on ovarian cells was dose-dependent. Adiponectin treatment significantly increased the expression of cyclin B in Caov3 and SKOV3, and reduced the levels of caspase-3 and bcl-2. Inhibitors of PI3K and MEK pathways significantly reduced the proliferation of attenuated Caov3 and SKOV3 by up-regulating cyclin B upon adiponectin treatment (p < 0.05), and thus alleviated the inhibitory effect of adiponectin on the expressions of caspase-3 and bcl-2.Conclusion: The findings demonstrate that adiponectin promotes proliferation of the cells via the PI3K/Akt and Raf/MEK/ERK pathways, and also provide new insights into ovarian cancer treatmment.Keywords: Adiponectin, Ovarian cancer, Proliferation, Apoptosis, PI3K/Akt pathwa

Low Cost Interconnected Architecture for the Hardware Spiking Neural Networks

A novel low cost interconnected architecture (LCIA) is proposed in this paper, which is an efficient solution for the neuron interconnections for the hardware spiking neural networks (SNNs). It is based on an all-to-all connection that takes each paired input and output nodes of multi-layer SNNs as the source and destination of connections. The aim is to maintain an efficient routing performance under low hardware overhead. A Networks-on-Chip (NoC) router is proposed as the fundamental component of the LCIA, where an effective scheduler is designed to address the traffic challenge due to irregular spikes. The router can find requests rapidly, make the arbitration decision promptly, and provide equal services to different network traffic requests. Experimental results show that the LCIA can manage the intercommunication of the multi-layer neural networks efficiently and have a low hardware overhead which can maintain the scalability of hardware SNNs

Antigenic analysis of classical swine fever virus E2 glycoprotein using pig antibodies identifies residues contributing to antigenic variation of the vaccine C-strain and group 2 strains circulating in China

BACKGROUND: Glycoprotein E2, the immunodominant protein of classical swine fever virus (CSFV), can induce neutralizing antibodies and confer protective immunity in pigs. Our previous phylogenetic analysis showed that subgroup 2.1 viruses branched away from subgroup 1.1, the vaccine C-strain lineage, and became dominant in China. The E2 glycoproteins of CSFV C-strain and recent subgroup 2.1 field isolates are genetically different. However, it has not been clearly demonstrated how this diversity affects antigenicity of the protein. RESULTS: Antigenic variation of glycoprotein E2 was observed not only between CSFV vaccine C-strain and subgroup 2.1 strains, but also among strains of the same subgroup 2.1 as determined by ELISA-based binding assay using pig antisera to the C-strain and a representative subgroup 2.1 strain QZ-07 currently circulating in China. Antigenic incompatibility of E2 proteins markedly reduced neutralization efficiency against heterologous strains. Single amino acid substitutions of D705N, L709P, G713E, N723S, and S779A on C-strain recombinant E2 (rE2) proteins significantly increased heterologous binding to anti-QZ-07 serum, suggesting that these residues may be responsible for the antigenic variation between the C-strain and subgroup 2.1 strains. Notably, a G713E substitution caused the most dramatic enhancement of binding of the variant C-strain rE2 protein to anti-QZ-07 serum. Multiple sequence alignment revealed that the glutamic acid residue at this position is conserved within group 2 strains, while the glycine residue is invariant among the vaccine strains, highlighting the role of the residue at this position as a major determinant of antigenic variation of E2. A variant Simpson's index analysis showed that both codons and amino acids of the residues contributing to antigenic variation have undergone similar diversification. CONCLUSIONS: These results demonstrate that CSFV vaccine C-strain and group 2 strains circulating in China differ in the antigenicity of their E2 glycoproteins. Systematic site-directed mutagenesis of the antigenic units has revealed residues that limit cross-reactivity. Our findings may be useful for the development of serological differential assays and improvement of immunogenicity of novel classical swine fever vaccines

ATG5 Promotes Death Signaling in Response to the Cyclic Depsipeptides Coibamide A and Apratoxin A

Our understanding of autophagy and lysosomal function has been greatly enhanced by the discovery of natural product structures that can serve as chemical probes to reveal new patterns of signal transduction in cells. Coibamide A is a cytotoxic marine natural product that induces mTOR-independent autophagy as an adaptive stress response that precedes cell death. Autophagy-related (ATG) protein 5 (ATG5) is required for coibamide-induced autophagy but not required for coibamide-induced apoptosis. Using wild-type and autophagy-deficient mouse embryonic fibroblasts (MEFs) we demonstrate that coibamide-induced toxicity is delayed in ATG5−/− cells relative to ATG5+/+ cells. Time-dependent changes in annexin V staining, membrane integrity, metabolic capacity and caspase activation indicated that MEFs with a functional autophagy pathway are more sensitive to coibamide A. This pattern could be distinguished from autophagy modulators that induce acute ER stress (thapsigargin, tunicamycin), ATP depletion (oligomycin A) or mTORC1 inhibition (rapamycin), but was shared with the Sec61 inhibitor apratoxin A. Coibamide- or apratoxin-induced cell stress was further distinguished from the action of thapsigargin by a pattern of early LC3-II accumulation in the absence of CHOP or BiP expression. Time-dependent changes in ATG5-ATG12, PARP1 and caspase-3 expression patterns were consistent with the conversion of ATG5 to a pro-death signal in response to both compounds

What motivates firms from emerging economies to go internationalization?

With advent of economic globalization, internationalization has become one of the most important strategies for firms to achieve sustainable growth. Based on the empirical research in the Yangtze River Delta region in China, the method of Correspondence Analysis was employed to study the motivations for going internationalization of Chinese enterprises. The main findings include: (1) the motivations for internationalization of enterprises depend on their scale, and largesized enterprises are mainly motivated by the purpose of creating global brands and enhancing domestic reputation; (2) the ownership of enterprises has obvious influence on their motivations for going internationalization, and (3) enterprises in different industries also show different levels of motivation for internationalization

Chronic Alcohol Causes Alteration of Lipidome Profiling in Brain

Much efforts have been tried to clarify the molecular mechanism of alcohol-induced brain damage from the perspective of genome and protein; however, the effect of chronic alcohol exposure on global lipid profiling of brain is unclear. In the present study, by using Q-TOF/MS-based lipidomics approach, we investigated the comprehensive lipidome profiling of brain from the rats orally administrated with alcohol daily, continuously for one year. Through systematically analysis of all lipids in prefrontal cortex (PFC) and striatum region, we found that long-term alcohol exposure profoundly modified brain lipidome profiling. Notably, three kinds of lipid classes, glycerophospholipid (GP), glycerolipid (GL) and fatty acyls (FA), were significantly increased in these two brain regions. Interestingly, most of the modified lipids were involved in synthetic pathways of endoplasmic reticulum (ER), which may result in ER stress-related metabolic disruption. Moreover, alcohol-modified lipid species displayed long length of carbon chain with high degree of unsaturation. Taken together, our results firstly present that chronic alcohol exposure markedly modifies brain lipidomic profiling, which may activate ER stress and eventually result in neurotoxicity. These findings provide a new insight into the mechanism of alcohol-related brain damage.Peer reviewe

Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61 alpha subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substratenonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61 alpha mutations identified from human HCT116 cells su ests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61 alpha mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61 alpha resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved druglike properties that are based on the coibamide pharmacophore.Peer reviewe

The MD Anderson prostate cancer patient-derived xenograft series (MDA PCa PDX) captures the molecular landscape of prostate cancer and facilitates marker-driven therapy development

BACKGROUND: Advances in prostate cancer (PCa) lag behind other tumor types partly due to the paucity of models reflecting key milestones in PCa progression. OBJECTIVE: To develop clinically relevant PCa models. DESIGN: Since 1996 we have generated clinically annotated patient-derived xenografts (PDXs) (the MDA PCa PDX series) linked to specific phenotypes reflecting all aspects of clinical PCa. RESULTS: We studied two cell line-derived xenografts and the first 80 PDXs derived from 47 human PCa donors. Of these, 47 PDXs derived from 22 donors are working models and can be expanded either as cell lines (MDA PCa 2a and 2b) or PDXs. The histopathologic, genomic, and molecular characteristics (AR, ERG, and PTEN loss) maintain fidelity with the human tumor and correlate with published findings. PDX growth response to mouse castration and targeted therapy illustrate their clinical utility. Comparative genomic hybridization and sequencing show significant differences in oncogenic pathways in pairs of PDXs derived from different areas of the same tumor. We also identified a recurrent focal deletion in an area that includes the SPOPL gene in PDXs derived from 7 human donors out of 28 studied (25%). SPOPL is a SPOP paralog, and SPOP mutations define a molecular subclass of PCa. SPOPL deletions are found in 7% of TCGA PCas, which suggests that our cohort is a reliable platform for targeted drug development. CONCLUSIONS: The MDA PCa PDX series is a dynamic resource that captures the molecular landscape of PCas progressing under novel treatments and enables optimization of PCa-specific, marker-driven therapy